P745: ASSESSMENT OF MIRNA BIOGENESIS GENES VARIANTS IN MDS DEVELOPMENT AND PROGRESSION
Authors: D. Bug, A. Tishkov, I. Moiseev, Y. Porozov, N. Petukhova
Journal: HemaSphere (2022)

Background: Experimental studies demonstrated that knockout of miRNA processing enzymes Dicer and Drosha is associated with the development of phenotype similar to myelodysplastic syndrome (MDS). DICER1 inherited syndrome is associated with cancer predisposition. There is a limited number of clinical studies evaluating the spectrum of mutations in the microRNA processing genes in patients with hematological malignancies and the pathogenic significance of these mutations is unknown. Thus, we conducted a bioinformatics analysis of sequencing data from our data set of MDS patients and available results in the public databases. Aims: We aim to identify DICER1 and DROSHA variants in hematological malignancies that lead to the protein function alteration based on bioinformatics analysis. Methods: Data on 35 previously sequenced high-risk MDS patients (Moiseev IS et al., PLoS One. 2021 Mar 17;16(3):e0248430) was analyzed, and the list of DICER1 and DROSHA variants was enriched with disease-specific coding variants retrieved from COSMIC database, and 3’-UTR-variants - from dbSNP. All collected variants were subjected to bioinformatics analysis. We used 3D-structures of Drosha and Dicer for molecular dynamics (MD) simulations for 200 and 300 ns, respectively (Schrödinger Suite 2020-4, Schrödinger, LLC, New York, NY, USA, 2020). The IntaRNA and MicroSNiPer algorithms were used to calculate interaction energies of 3’-UTRs hybridizing with the most abundant miRNAs in MSCs to evaluate noncoding variants. Results: The prevalence of mutations in DICER1 and DROSHA was 54% and 17%, respectively, predominantly located in 5’-UTR and 3’-UTR regions. Detected allele frequencies and prevalence suggested their association with minor clones within the bone marrow niche. MD simulation demonstrated apparent structural alterations with consequent protein dysfunction. RMSD results and calculated total energy of studied proteins were higher for all mutants compared to wild-type Drosha (at least 30% RMSD shift). All mutations were shown to be located in close proximity of the miRNA binding site, all were destabilizing for protein structure alone with consequent alterations in bonds and protein-miRNA binding. The most significant structural shift and protein destabilization was detected for DICER1 variants F508C and T993R characterized by local transformation of bonds interplay. The conformational changes in the protein due to Dicer variants were studied in 300 ns: differences in average RMSD values were within ~ 1Å between WT protein and mutants. The most significant changes were observed at the local area for I445S, F508C, and T993R characterized by shift of molecular bonds and alteration of protein stability parameters. A list of SNPs that exhibit the highest repression of DICER1 and DROSHA was obtained (Table 1). The most prominent SNPs (rs1479981622, rs536092006, rs1296755923, rs1207839989) had less than 0.03% population allele frequency which is in concordance with their possible pathogenicity. Image:Summary/Conclusion: We identified potential coding and non-coding “hot spots” for future analysis. Also the study shows that 3’-UTR SNPs of DICER1, DROSHA have more potential to impact MDS progression than coding variants. Simultaneously, we found low frequency of reports for these mutations in the available databases, indicating bias and absence of these genes in the targeted sequencing panels. This builds the foundation for future research of DICER1 and DROSHA clinical role in hematological malignancies.